FRMD4A regulates the entry of west nile virus into glioblastoma cells

FRMD4A REGULATES THE ENTRY OF
WEST NILE VIRUS INTO GLIOBLASTOMA CELLS

PANG JUNXIONG, VINCENT

NATIONAL UNIVERSITY OF SINGAPORE

2009


FRMD4A REGULATES THE ENTRY OF
WEST NILE VIRUS INTO GLIOBLASTOMA CELLS

PANG JUNXIONG, VINCENT
[B.Sc. (Hons.), NUS]

A THESIS SUBMITTED

FOR THE DEGREE OF MASTER OF SCIENCE IN
INFECTIOUS DISEASES, VACCINOLOGY AND
DRUG DISCOVERY

DEPARTMENT OF MICROBIOLOGY

NATIONAL UNIVERSITY OF SINGAPORE
2009


MATERIALS FROM THIS STUDY HAVE BEEN PRESENTED
AT THE FOLLOWING CONFERENCE

JX Pang and ML Ng. (2008). FRMD4A, a FERM domain-containing gene, regulates
the permissivity of A172 glioblastoma cells towards West Nile virus infection. 9th
Asia Pacific Microscopy Conference (APMC9). Jeju, Korea. (Oral presentation)
(APMC9 travel scholarship)

i


ACKNOWLEDGEMENTS
I would like to express my sincere thanks and gratitude to the following people for
their contributions during this one year of fruitful research:

Professor Mary Ng– For her supportive supervision and steadfast guidance, and for
spending her weekends reviewing this thesis.

Madam Loy Boon Pheng– For her efficient running of the laboratory, and her
professionalism in maintaining the logistical and safety issues in the laboratory.

Terence Tan, Bhuvana, Yeo Kim Long, Chong Munkeat, Edwin, Adrian, Melvin,
Anthony, Su Min and Li Shan– For their generous advice and support, and also for
their Flavivirology spirit and fun in the laboratory.

Mr. Clement Khaw (Nikon Imaging Centre, Biopolis)– For his prompt expert advice
on confocal microscopy imaging services.

My wife, Xiaoman, all family members and friends– For their emotional support and
encouragements.

ii

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ....................................................................................... ii
TABLE OF CONTENTS .......................................................................................... iii
LIST OF TABLES…………………………………………………………………..vi
LIST OF FIGURES…………………………………………………………………vi
SUMMARY .............................................................................................................. viii
INTRODUCTION…………………………………………………………………...1
1.0.

LITERATURE REVIEW…………………………………………………….3

1.1.

HISTORY OF WEST NILE VIRUS………………………………………………..3

1.2.

EPIDEMIOLOGY OF WEST NILE VIRUS INFECTION……………………............3

1.3.

CLINICAL MANIFESTATIONS OF WEST NILE VIRUS INFECTION……………...7

1.4.

VIRUS MORPHOLOGY...………………………………………………………..8

1.5.

VIRUS ENTRY, ASSEMBLY AND MATURATION………………………………...9

1.6.

VIRUS-HOST INTERACTIONS...……………………………………………….14

1.7.

THE FERM DOMAIN SUPERFAMILY…………………………………………..18

1.8.

GENE SILENCING WITH MICRORNA...………………………………………19

1.9.

OBJECTIVES OF STUDY...……………………………………………………..21

2.0.

MATERIALS AND METHODS……………………………………………22

2.1. CELL CULTURE…………………………………………………………………22
2.1.1. Cell Lines...……………………………………………………………..22
2.1.3. Media for Cell Culture…...……………………………………………..23
2.1.4. Regeneration, Cultivation and Propagation of Cell Lines………………23
2.2. INFECTION OF CELLS…………………………………………………………..25
2.2.1. Virus Strains…………………………………………………………….25
2.2.2. Infection of Cell Monolayers and Production of Virus Pool…………...25
2.2.3. Plaque Assay……………………………………………………………26

2.3. MICROSCOPY…………………………………………………………………..27
2.3.1. Light Microscopy………………………………………………………..27
2.3.2. Indirect Immunofluorescence Microscopy …………………….……….27
iii


2.4. MOLECULAR BIOLOGY TECHNIQUES………………………………………….30
2.4.1. Total RNA Isolation from Cell Culture…………………………………30
2.4.2. Small Scale Purification and Screening of Plasmid DNA……………...31
2.4.3. RNA and DNA Plasmid Quantification and Quality Determination…...31
2.4.4. Determination of RNA and DNA Plasmid Integrity…..………………..32
2.4.5. Automatic DNA sequencing……………………………………………32
2.4.6. Western Blot……………………………………………………………33
2.5. SEMI-QUANTITATIVE REVERSE TRANSCRIPTION

AND

QUANTITATIVE REAL-

TIME PCR……………………………………………………………………………35
2.5.1. Synthesis of Oligonucleotides…………………………………………..35
2.5.2. Semi-Quantitative Reverse Transcription PCR………………………...35
2.5.3. Real-Time PCR…………………………………………………………36
2.6. GENE SILENCING WITH MICRORNA (MIRNA)………………………………..37
2.6.1. Generation of pcDNATM 6.2-GW/miR expression clone………………37
2.6.2. Transient Silencing of FRMD4A & INDO in A172 cells………………38
2.7. CLONING OF FULL-LENGTH FRMD4A AND TRUNCATED FRMD4A…………38
2.7.1. First strand cDNA synthesis……………………………………………38
2.7.2.

PCR

Amplification

of

Full-Length

and

Partial

Fragments

of

FRMD4A……………………………………………………………………….40
2.7.3. Cloning of FERM domain into GFP Vector……………………………41
2.8. BIOINFORMATIC ANALYSES……………………………………………………42
3.0. RESULTS……………………………………………………………………...43
3.1. VALIDATION OF MICROARRAY ANALYSIS OF FRMD4A AND INDO………...43
3.1.1. Total RNA Integrity and Purity………………………………………...45
3.1.2. Primer Specificity of FRMD4A AND INDO …………………………....46
3.1.3. Endogenous Control Assessment ………………………………………46
3.1.4. Semi-Quantitative RT-PCR ……………………………………………48
3.1.5. Real Time PCR Analyses ………………………………………………50
3.2. IMPACT OF SILENCING FRMD4A AND INDO ON WNV INFECTION………….54
3.2.1. Construction of FRMD4A-and INDO-Silencing Plasmid………………54
3.2.2. Transient Silencing Analyses of FRMD4A in A172 cells and its impact on
virus infection…………………………………………………………………..58

iv


3.2.3. Transient Silencing Analyses of INDO in A172 cells and its impact on
virus infection ………………………………………………………………….59
3.3. ELUCIDATION
LESS PERMISSIVE

OF THE

ROLE

A172 CELLS

TO

OF

FRMD4A

AND ITS

WNV INFECTION

FERM DOMAIN

WITH

IN THE

BIOINFORMATICS

AND

IMMUNOFLUORESCENCE MICROSCOPY…………………………………………….61
3.3.1. Bioinformatics Analyses of FRMD4A………………………………….61
3.3.2. Cloning of Full-Length FRMD4A and its FERM Domain……………..65
3.3.3. Colocalisation of WNV and Integrins………………………………….67
3.3.4. No Colocalisation between FERM Domain of FRMD4A and Actin
Filaments………………………………………………………………………..67
3.3.5. Colocalisation of FERM Domain of FRMD4A and Integrins………….70
3.3.6. Colocalisation of FERM Domain of FRMD4A and WNV…………….71
3.3.7. FERM Domain may Regulate the Level of Phosphorylation of FAK
Tyrosine 397……………………………………………………………………73
4.0. DISCUSSION & CONCLUSION……………………………………………77
REFERENCES……………………………………………………………………...84
APPENDIX 1: Media for Tissue Culture of Cell Lines……………………………100
APPENDIX 2: Reagents for Subculturing of Cells………………………………..102
APPENDIX 3: Reagents for Infection of Cells & Plaque Assays…………………104
APPENDIX 4: Reagents for Indirect Immunofluorescence Microscopy………….105
APPENDIX 5: Reagents for Molecular Biology Techniques……………………..106
APPENDIX 6: List of Oligonucleotides…………………………………………...110

v


LIST OF TABLES
2.0

MATERIALS AND METHODS

2-1

Antibodies and their working dilution used in IFA………………….

28

LIST OF FIGURES
1.0

LITERATURE REVIEW

1-1

Epidemics caused by West Nile virus, 1937–2006………………..

4

1-2

Phylogenetic tree of West Nile viruses based on the sequence of the 6
envelope protein………………………….................................

1-3

The immature and mature flavivirus virion………………………. 9

1-4

Structural arrangement of flavivirus envelope protein…………

9

1-5

The Flavivirus replication cycle………………………………

10

1-6

Proposed rearrangement of the E proteins during maturation 12
and fusion………………………................................................

3.0

RESULTS

3-1

Differential WNV infection in selected cells …………………….

44

3-2

Integrity and purity assessment of extracted total RNA …………

45

3-3

Primer specificity of FRMD4A and INDO primers.………………

46

3-4

Endogenous control assessment for real-time PCR ……………....

47

3-5

Semi-quantitative RT-PCR of FRMD4A and INDO ……………..

49

3-6

Dissociation curve of FRMD4A (A) and INDO ………………….

51

3-7

Real-time PCR analyses of FRMD4A and INDO in WNV-infected

52

A172 and HeLa cells ……………………………………………..
3-8

Real-time PCR analysis of FRMD4A and INDO mRNA expression 53
level (Ct value) in A172 cells and HeLa cells…………

3-9

Relative fold change of FRMD4A and INDO between WNV-infected 53
A172 and HeLa cells using real-time PCR ……………...

3-10

Schematic diagrams of FRMD4A (A) and INDO (B) mRNA, and 56
vi


their respective miRNA sequence sites.………………………….
3-11

Generation of double-stranded (ds) oligo (A) and pre-miRNA- 57
expressing vector for silencing …………………………………..

3-12

Transient silencing of FRMD4A in A172 cells. …..........................

58

3-13

The impact of transient silencing FRMD4A on virus titre in A172 59
cells ……………………………………………………………….

3-14

Transient silencing of INDO in A172 cells ……………………….

60

3-15

The impact of transient silencing INDO on virus titre in A172 cells 61
…………….............................................................................

3-16

Conserved domains of FRMD4A …………………………………

61

3-17

Amino acid sequence homology of FERM domain compared with

62

that of erythroid protein 4.1……………………………………….
3-18

Clustering of the FERM domain of RADIXIN, FRMD4A, TALIN

64

and FAK ……………………………..............................................
3-19

Cloning of full-length and FERM domain of FRMD4A…………..

66

3-20

Immunofluorescence microscopy images of integrin (B) and WNV

69

(C) association in WNV-infected A172 cells ……………..
3-21

Immunofluorescence microscopy images of FERM-GFP and actin

70

association ………………………………………………………...
3-22

Immunofluorescence microscopy images of FERM-GFP and integrin 72
association in mock-infected and infected A172 cells……

3-23

Immunofluorescence microscopy images of FERM-GFP and WNV 73
association. Nuclei staining with DAPI …………................

3-24

Phosphorylation of tyrosine 397 of Focal Adhesion Kinase (FAK) in

75

WNV-infected A172 cells ……………………………………...
3-25

Semi-quantitation of FAK tyrosine 397 phosphorylation in the 76
following cells …………………………………………………….

4.0
4-1

DISCUSSION
A cartoon of the proposed mechanism that regulates the WNV entry

81

in WNV-infected A172……………………………...............

vii

____________________________________________________

__Summary

SUMMARY
West Nile virus (WNV) is a mosquito-borne flavivirus. It can cause fatal
meningoencephalitis

in

infected

victims

especially

in

elderly

and

immunocompromised. This re-emerging virus has recently caused large epidemics in
the Western Hemisphere. Despite advances in WNV research, the mechanism of its
molecular pathogenesis is still not well understood. It has also been shown that
different cell types have different permissivity to WNV infection. Differential
permissivity could be one of the factors that contribute to different degree of
pathogenesis. Hence, by exploring the transcriptome profile of two different cells with
differential permissivity, a better understanding of the molecular pathogenesis of
WNV could be attained.

The initial studies on different human host cells have found that A172 cells
(glioblastoma) were not as permissive as HeLa cells (cervical adenocarcinoma) to
WNV (Sarafend) infection. Based on the results of a previous study by Koh and Ng
(2005) on the global transcriptome profiles of these two different host cells,
differentially expressed FRMD4A and INDO were selected as the genes of interest.
The gene expression profile of FRMD4A and INDO were further validated by reversetranscription and real-time polymerase chain reaction (PCR). Silencing of FRMD4A
and INDO in A172 cells showed ten-fold increase and no increase in virus titre,
respectively. Hence, INDO was dropped out as it showed no anti-viral role and
FRMD4A was chosen for further research. It was also observed that FRMD4A only
expressed in A172 cells and not HeLa cells. This showed that FRMD4A is an antiviral host factor that can resist WNV infection, found only in A172 cells.

viii


____________________________________________________

__Summary

Based on indirect immunofluorescence confocal microscopy, FRMD4A was
observed to interact with the activated αvβ3 integrin via the FERM domain at the Nterminal of FRMD4A protein. Activated αvβ3 integrin have been shown previously to
mediate WNV entry via the activated focal adhesion kinase (FAK) pathway. Through
bioinformatics analyses, it was observed that FERM domain of FRMD4A may
compete with FAK binding event to the activated αvβ3 integrin. As a result, the level
of phosphorylation of FAK was affected that might have hindered the entry of WNV.
Hence, this study provided insights into how FRMD4A regulates the entry of WNV
via the activated αvβ3 integrin pathway in A172 cells, making them less permissive to
WNV infection. The entry event is often a major determinant of virus tropism and
pathogenesis (Schneider-Schaulies, 2000). Understanding this early event of virus
infection in more details will provide opportunities to develop strategies to reduce the

burden of WNV infection.

ix


Introduction

INTRODUCTION
The completion of the Human Genome Project has revolutionised biomedical
sciences gradually towards functional genomics. Functional genomics involve the
analyses and understanding of many genes (and proteins) functions and their
interactions simultaneously. As a result, an overall biological mechanism of how
certain phenotypes arise can be proposed and this can enhance the progress of drug
discovery and vaccine developments against the emerging infectious diseases.

Techniques of functional genomics include high-throughput methods for gene
expression profiling at the transcript and protein levels, and the application of
bioinformatics. DNA microarray and two-dimensional gel electrophoresis are the
common methods for gene expression profiling at the transcript and protein level,
respectively. Both DNA microarrays and proteomics hold great promise for the study
of complex biological systems with applications in molecular medicine (Celis et al.,
2000). A vast amount of gene and protein expression data is usually generated and
these data may provide information in understanding the regulatory events involved in
normal and diseased processes.

Flaviviruses are emerging pathogens of increasingly important public health
concern in the world. For some flaviviruses such as West Nile virus (WNV), although
much has been learned about their molecular biology, neither effective vaccine nor
antiviral therapy is available yet. In order to generate an effective vaccine, the vaccine
must be immunogenic enough to generate an effective humoral immune response,

producing neutralising antibodies but not too reactogenic that it is harmful to the host.
In addition, an effective vaccine has to provide protection against all different
1


____________________________________________________

__Summary

serotypes and strains of the virus. As such, even though the development of safe and
effective vaccines remains to be critical for controlling the disease in the long run,
alternatively, antiviral therapy is an approach to be developed in parallel as well

Since WNV alternates between insect vectors and vertebrates in nature, any
cellular proteins that this virus uses during replication would be expected to be
evolutionarily conserved. Of particular interest will be the identification of cell
protein(s) used for virus attachment and entry, and elucidation of molecular
mechanisms involved in virus replication. Viruses use cell proteins during many
stages of their replication cycles, including attachment, entry, translation,
transcription/replication, and assembly. Viruses also interact with cell proteins to alter
the intracellular environment or cell architecture so that it is more favourable for virus
replication. The replication can also inactivate intracellular defence mechanisms, such
as apoptosis and interferon pathways. Mutations in cell proteins involved can cause
disruptions of these critical virus-host interactions. These virus-host interactions may
thus represent novel targets for the development of new anti-viral agents.

A DNA microarray genomic study was carried out previously by Koh and Ng
(2005) to elucidate host factors involved in the different permissiveness of HeLa and
A172 cell lines to WNV (Sarafend) infection. Based on the findings, an attempt was
therefore made to further investigate whether any of these differentially expressed

host factors play a role in anti-viral mechanism in A172 cells as it may be one the
factors that caused brain inflammation. This host factor may also represent novel
target for the development of new anti-viral agents.

2


Literature Review

CHAPTER 1
LITERATURE REVIEW

1.1.

History of West Nile Virus
West Nile virus (WNV) was first isolated in 1937 from the blood of a febrile

adult woman participating in a malaria study in the West Nile region of Uganda
(Smithburn et al., 1940). Before the fall of 1999, WNV was considered to be
relatively unimportant as a human and animal pathogen and it was classified under the
genus Flavivirus under the family Flaviviridae by a cross-neutralisation test (Calisher
et al., 1989). It is a member of the Japanese encephalitis virus serogroup of
flaviviruses, which includes a number of closely related viruses that also cause human
disease, including Japanese encephalitis virus (JEV) in Asia, St. Louis encephalitis
virus (SLEV) in the Americas, and Murray Valley encephalitis virus (MVEV) in
Australia (Mackenzie et al., 2002; Gubler et al., 2007). These viruses have a similar
transmission cycle, with broad vector range such as Culex species mosquitoes serving
as the enzootic and/ or epizootic vectors and broad vertebrate host range such as birds
serving as the natural vertebrate host, humans and domestic animals, such as horses,
are generally thought to be incidental hosts.

1.2. Epidemiology of West Nile Virus Infection
From 1937 to 1999, epidemic of infection only occurred occasionally
(Romania and Morocco in 1996; Tunisia in 1997; Italy in 1998; Figure 1-1) and
infection of human, birds and horses were generally asymptomatic or mild. In
3


________________________________________________

Literature Review

addition, neurologic disease and death were very uncommon (Murgue et al., 2001;
Murgue et al., 2002; Hurlburt et al., 1956).

Figure 1-1. Epidemics caused by West Nile virus, 1937–2006. The red stars indicate epidemics that
have occurred since 1994 that have been associated with severe and fatal neurologic disease in humans,
birds, and/or equines (adapted from Gubler DJ, 2007).

In 1999, an epidemic of WNV infection occurred in some parts of United
States such as New York, Connecticut, and New Jersey (Hayes et al., 2006) and the
severity of the disease was seen to increase amongst those who developed clinical
symptoms (Petersen and Roehrig, 2001). This WNV outbreak was suggested to be
due to the introduction of WNV in spring or early summer of 1999 by an infected
human arriving from Israel, which was also facing WNV epidemic in Tel Aviv at that
time (Giladi et al., 2001). In addition, it was found that the epidemic was due to the
emergence of a new variant of WNV designated “Isr98/NY99” (Lanciotti et al.,
2002). This strain is characterized by a high avian death rate and an apparent increase
in human disease severity as it moved westward of United States (Solomon and
Winter, 2004). This was consistent with the hypothesis that there were some changes

in the neurovirulent properties of the virus (Ceccaldi et al., 2004).

4


Literature Review
From 1999, there were increasing number of cases with neuroinvasive disease
and death (Gubler, 2007). This is likely due to the increasing numbers of migratory
birds that fly south to Central and South America in the fall and back north to the
United States and Canada along specific flyways in the spring (Gubler, 2007). These
migratory birds presented an increased risk of spreading WNV, resulting in the
increasing number of cases. West Nile virus infection was observed via several novel
modalities of transmission to humans besides advances in transportation and
globalisation. These include transplacental transmission to the foetus, transmission via
breast milk, blood transfusion, or laboratory contamination through percutaneous
inoculation (Peterson and Roehrig, 2001; Hayes and O’Leary, 2004).

Wild bird species develop high levels of viremia after WNV infection and are
able to sustain viremic levels of WNV of at least 105 PFU/ml of serum (the minimum
level estimated to be required to infect a feeding mosquito) for days to weeks. They
are the main reservoir hosts in endemic regions for the virus, which can initiate
epizootics outside the endemic areas (Bernard et al., 2001; Petersen and Roehrig,
2001).

West Nile virus has been isolated from Culex, Aedes, Anopheles, Minomyia,
and Mansonia mosquitoes in Africa, Asia, and the United States, but Culex species
are the most susceptible to WNV infection (Burke and Monath, 2001; Ilkal et al.,
1997). Culex mosquitoes feed on infected wild bird species. This increases the
possibility of vertical transmission from mosquito to eggs since infected wild birds
can have high levels of viremia (Turell et al., 2000). Natural vertical transmission of

WNV in Culex mosquitoes in Africa has been reported and is expected to enhance
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________________________________________________

Literature Review

virus maintenance in nature (Miller et al., 2000). Humans and horses are incidental
hosts with low viremic levels and it is still unknown what roles they play in the
transmission cycle of WNV (Gubler, 2007).

The existing WNV isolates are grouped into two genetic Lineages (1 and 2) on
the basis of signature amino acid substitutions or deletions in their envelope proteins
(Berthet et al., 1997). Due to antigenic cross-reactivity between different flaviviruses,
techniques such as in situ hybridization and sequence analyses of real-time
polymerase chain reaction (PCR) products are required to unequivocally identify
WNV as the causative agent in infections (Briese et al., 2002; Lanciotti et al., 2002).
All members belong to the same clade share more than or equal to 98% homology
with each other (Figure 1-2), thus suggesting that they all had a common ancestor. All
WNV isolates that are associated with human diseases are found in Lineage 1, while
Lineage 2 viruses are mainly restricted to endemic enzootic infection in Africa (Jia et
al., 1999; Lanciotti et al., 2002).

Figure 1-2 Phylogenetic tree of West Nile viruses based on sequence of the envelope gene. Viruses
were isolated during the epidemics indicated by red stars in Figure 1-1, all of which belong to the same
clade, suggesting a common origin. Figure appears courtesy of the Centers for Disease Control and
Prevention (adapted from Gubler DJ, 2007)

6

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Literature Review

1.3. Clinical Manifestations of West Nile Virus Infection
According to the Centre for Disease Control and Prevention (CDC), WNV
infections may be asymptomatic or may result in illnesses of variable severity
sometimes associated with central nervous system (CNS) involvement. West Nile
Fever (WNF) is the most common symptom observed in humans. The course of the
fever is sometimes biphasic, and a rash on the chest, back, and upper extremities often
develops during or just after the fever (Burke and Monath, 2001). When the CNS is
affected, clinical syndromes ranging from febrile headache to aseptic meningitis to
encephalitis may occur (Omalu et al., 2003, Briese et al., 2000), and these are usually
indistinguishable from similar syndromes caused by other arboviruses, and hence,
may lead to misdiagnosis. The brainstem, particularly the medulla, is the primary
central nervous system (CNS) target. Humans aged 60 and older have an increased
risk of developing this fatal disease (Sampson et al., 2000; Chowers et al., 2001).
WNV meningitis is characterized by fever, headache, stiff neck, and pleocytosis.
WNV encephalitis is characterized by fever, headache, and altered mental status
ranging from confusion to coma with or without additional signs of brain dysfunction
(e.g., paresis or paralysis, cranial nerve palsies, sensory deficits, abnormal reflexes,
generalized convulsions, and abnormal movements). Flacid paralysis and muscle
weakness, similar to polio-like syndrome, have also been reported in the absence of
fever or meningo-encephalitis (Li et al., 2003; Arturo et al., 2003).

Histopathological studies revealed that, WNV could be detected but in
different viral titres in all major organs such as liver, kidney, heart and spleen, and in
most part of the brain (88%), including glial cells and neurons (Steele et al., 2000).

Neuropathogenicity was also observed in infected animals whereby it is similar to
7


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Literature Review

poliomyeloencephalitis. It was characterized by T-lymphocytes and, to a lesser extent,
macrophage infiltration within the CNS, with multifocal glial nodules and some
nueronophagia (Cantile et al., 2001). As high levels of WNV-reactive serum IgM
antibodies could still be detected in confirmed human cases (Roehrig et al., 2003) and
in animal studies (Xiao et al., 2001) of WNV encephalitis as long as 1.5 years after
onset, there is a possibility of viral persistence within the CNS.

1.4. Virus Morphology
West Nile virus belongs to the family Flavivirdae. The virions are small
(~50nm in diameter), spherical, enveloped, and have a buoyant density of ~1.2g/cm3.
The WNV genome is a single-stranded RNA of positive polarity (mRNA sense) and
is 11,029 bases in length, containing a single open reading frame (ORF) of 10,301
bases. The virus contain three structural proteins which include the majority of
flavivirus antigenic and functional determinants (Heinz and Roehrig, 1990): a
nucleocapsid protein (C protein, 14kDa), a lipid membrane protein (M protein, 8kDa),
and a large envelope glycoprotein (E protein, 55kDa). Figure 1-3 shows the structure
of the virus particle and Figure 1-4 shows the structural arrangement of the envelope
proteins. The E glycoprotein is the principal stimulus for the development of
neutralizing antibodies and it contains a fusion peptide responsible for inserting the
virus into the host cell membrane. Generally, the E proteins of most flaviviruses are
glycosylated, and the glycosylation of certain amino acid residues have been
postulated to contribute to the pathogenicity of the virus (Beasley et al., 2004). Hence,

varying N-glycosylation sites could also be important in epitope definition (Seligman
and Bucher, 2003).
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Literature Review

Figure 1-3. The immature and
mature flavivirus virions. The
heterodimers of prM and E are
shown on the left (immature
virion) and the homodimers of E,
following cleavage of prM, on the
right (mature virion). The
icosahedral nucleocapsid consists
of viral C protein and genomic
RNA, and is surrounded by a lipid
bilayer in which the viral E and
prM/M proteins are embedded.
Viral maturation is triggered by
the cleavage of prM to pr and M
proteins by the host protease furin
(adapted from Shi, 2002).

Figure
1-4.
Structural
arrangement

of
flavivirus
envelope protein. Diagrams of
the flavivirus ectodomain and
transmembrane domain proteins
side and top views. The stem and
transmembrane helices of the E
(E-H1, E-H2, E-T1 and E-T2) and
M (M-H, M-T1 and M-T2)
proteins are shown in blue and
orange,
respectively.
The
conserved amino acid sequence of
the region between the two E
protein stem helices is marked CS
(adapted from Mukhopadhyay et
al., 2005).

1.5. Virus Entry, Assembly and Maturation
WNV replicates in a wide variety of cell cultures, including primary chicken,
duck and mouse embryo cells and continuous cell lines from monkeys, humans, pigs,
rodents, amphibians, and insects, but does not cause obvious cytopathology in many
cell lines (Brinton, 1986). It was demonstrated that although embryonic stem (ES)
cells were relatively resistant to WNV infection before differentiation, they became
permissive to WNV infection once differentiated, and die by the process of apoptosis
(Shrestha et al., 2003). Since flaviviruses are transmitted between insect and
9

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Literature Review

vertebrate hosts during their natural transmission cycle, it is likely that the cell
receptor(s) they utilize to gain entry into the cells is a highly conserved protein
(Brinton, 2002). The receptor for WNV (Sarafend) was found to be a 105-kDa
protease-sensitive, N-linked glycoprotein in Vero and murine neuroblastoma 2A cells
(Chu and Ng, 2003a). Subsequently, it was determined to be the αVβ3-integrin
receptor (Chu and Ng, 2004b). Alternatively, WNV entry can be independent of αVβ3integrin receptor. The virus was shown to enter via cholesterol-rich membrane
microdomain (Medigeshi et al., 2008)

Figure 1-5. The Flavivirus replication cycle. Virions attach to the surface of a host cell and
subsequently enter the cell by receptor-mediated endocytosis (see Figure). Several primary receptors
and low-affinity co-receptors for flaviviruses have been identified. Acidification of the endosomal
vesicle triggers conformational changes in the virion, resulting in fusion of the viral and lysosomal
membranes, and particle disassembly. Once the genome is released into the cytoplasm, the positivesense RNA is translated into a single polyprotein that is processed co- and post-translationally by viral
and host proteases. Genome replication occurs on intracellular membranes. Virus assembly occurs on
the surface of the endoplasmic reticulum (ER) when the structural proteins and newly synthesized RNA
buds into the lumen of the ER. The resultant non-infectious, immature viral and subviral particles are
transported through the trans-Golgi network (TGN). The immature virion particles are cleaved by the
host protease furin, resulting in mature, infectious particles. Subviral particles are also cleaved by furin.
Mature virions and subviral particles are subsequently released by exocytosis (adapted from
Mukhopadhyay et al., 2005).

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The pathway for flavivirus entry into host cells is through clathrin-mediated
endocytosis, which is triggered by an internalization signal (di-leucine or YXXΦ) in
the cytoplasmic tail of the receptor (Chu and Ng, 2004a). Clathrin is assembled on the
inside face of the plasma membrane to form an electron dense coat known as clathrincoated pit. Clathrin interacts with a number of accessory protein molecules (Eps15,
ampiphysin and AP2 adapter protein) as well as the dynamin GTPase which is
responsible for releasing the internalized vesicle from the plasma membrane (Marsh
and McMahon, 1999).

This is followed by low-pH fusion of the viral membrane with the lysosomal
vesicle membrane, releasing the nucleocapsid into the cytoplasm [(Heinz and Allison,
2000) (Figure 1-5 and 1-6)]. The reduced pH causes the conformational
rearrangement of the E proteins, allowing the interactions of the virus E proteins with
the lysosomal membrane to form hemifusion pores for the release of viral
nucleocapsids into the cytoplasm for uncoating and replication (Modis et al., 2004).

The RNA genome is released and translated into a single polyprotein (Figure
1-5). The viral serine protease, NS2B-NS3, and several cell proteases then cleave the
polyprotein at multiple sites to generate the mature viral proteins (Figure 1-5). The
viral RNA-dependent RNA polymerase (RdRp), NS5, in conjunction with other viral
nonstructural proteins and possibly cell proteins, copies complementary minus strands
from the genomic RNA template, and these minus-strand RNAs in turn serve as
templates for the synthesis of new genomic RNAs. Upon WNV infection, extensive
reorganization and proliferation of both smooth and rough endoplasmic reticula were
observed (Ko et al., 1979; Murphy, 1980; Westaway and Ng, 1980; Lindenbach and
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Rice, 1999). There were also induction of unique sets of membranous structures, but
their functions during infection mostly remained elusive (Westaway et al., 2002).
One of such generic flavivirus-induced features, in both vertebrate and invertebrate
cells, is the formation of vesicles packets that contains bi-layered membrane vesicles
of 50-100 nm in size. These vesicles enclosed distinctively single or double-stranded
‘thread-like’ structures during early stages of infection (Ng, 1987).

Figure 1-6 Proposed rearrangement of the E proteins during maturation and fusion. a The E
proteins in the immature virus (left) rearrange to form the mature virus particle (right). b The E protein
dimers in the mature virus (left) are shown undergoing a rearrangement to form the putative T=3
fusogenic intermediate structure (right) with a possible intermediate (centre). The arrows indicate the
direction of the E rotation. The solid triangle indicates the position of a quasi three-fold axis. This
suggested rearrangement would require a ~10% radial expansion of the particle between the
intermediate (centre) and fusogenic form (right) (adapted from Mukhopadhyay et al., 2005).

Flavivirus assembly occurs in association with the ER membranes (Figure 15). Intracellular immature virions, which contain heterodimers of E and prM proteins,
accumulate in vesicles and are then transported through the host secretory pathway
(Heinz et al., 1994). It has been shown by electron microscopy that mature virions can
be found within the lumen of endoplasmic reticulum (Matsumura et al., 1977;
Sriurairatna and Bhamarapravati, 1977; Hase et al., 1989; Ng, 1987) at the perinuclear
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area of the cytoplasm (Murphy, 1980; Westaway and Ng, 1980). The glycosylated
and hydrophilic N-terminal portion of prM protein is cleaved in the trans-Golgi
network by cellular furin or a related protease (Stadler et al., 1997). The C-terminal
portion (M) remains inserted in the envelope protein of the mature virion (Murray et
al., 1993). The prM-E proteins interaction may maintain the E protein in a stable,
fusion-inactive conformation during the assembly and release of new virions (Heinz
and Allison, 2000). Recently, it has been shown that the pr peptide beta-barrel
structure of immature virus at neutral pH covers the fusion loop in E protein,
preventing fusion with host cell membranes (Li et al., 2008). Virus maturation
involves 60 trimers of prM-E proteins heterodimers that project from the virus surface
to dissociate and form 90 E protein homodimers, which lie flat on the virus surface.
During fusion with host cell, the anti-parallel E protein homodimers dissociate into
monomers, which then reassociate into parallel homotrimers (Figure 1-6)
(Mukhopadhyay et al., 2005).

Assembly of WN (Sarafend) virus is, however, slightly different from the
process shown above, which is generally true for other flaviviruses. With the use of
cryo-immunoelectron microscopy, the precursor of nucleocapsid particles from WNV
was observed to be closely associated with the envelope proteins at the host cell’s
plasma membrane (Ng et al., 2001). Instead of maturing within the endoplasmic
reticulum, WNV was found to mature (cis-mode) at the plasma membrane (Ng et al.,
1994). This contrasts with the trans-mode of maturation observed for most flavivirus
where mature virus particles are released from cells by exocytosis (Mason, 1989;
Nowak et al., 1989).

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Egress of WNV had been observed to occur predominantly at the apical
surface of polarized Vero cells, suggesting the involvement of a microtubuledependent, polarized sorting mechanism for WNV proteins (Chu and Ng, 2002a).
Previous study has shown that both E and C proteins were strongly associated and
transported along the microtubules to the plasma membrane for assembly (Chu and
Ng, 2002b). It was also observed in the same study that the association of E protein
and microtubules was sensitive to high salt extraction but resistant to Triton X-100
and octyl glycoside extraction. This suggested that virus E protein and possibly also C
protein associate effectively with the microtubules through an ionic interaction (Chu
and Ng, 2002b).

1.6. Virus-Host Interactions
Infection and replication of viruses in vertebrate cells resulted in the alteration
of expression of many cellular genes and these differentially expressed genes can be
identified using a variety of techniques such as high-density DNA microarrays,
differential display or subtraction hybridization (Manger and Relman, 2000). Such
changes in host gene expression could be a cellular antivirus response, a virusinduced response that is beneficial or even essential for virus survival, or a nonspecific response that neither promotes nor prevents virus infection (Saha and
Rangarajan, 2003). In addition, some cell types may response differently to WNV
infection (Silva et al., 2007) and this make the study of WNV pathogenesis more
complicated but still essential so as to develop an effective antiviral strategy.
Infection of diploid vertebrate cells with WNV has been reported to increase
cell surface expression of MHC-1, which was activated by NF-κB (Kesson and King,
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